Answer to Question 1 of 1995

This question was written with the intent of making the following answer obvious, if you understood the methods involved:

Judy suggested that Jennifer repeat the experiment, this time using the T3 RNA polymerase.

The above single sentence, or a variation thereof, would have been sufficient to answer this question and would have received full credit. Additional detail was not necessary. In general, it's good to try to answer exam questions as briefly and directly as possible. Brevity saves you time and saves the instructor from eyestrain. In addition, if you write more detail than necessary, the instructor may find that some of the additional, unnecessary material is incorrect, and he/she may reduce your credit.

To answer this question, it is important to realize that the DNA isolated from the M13 vector containing the IMPORTANT gene would be single-stranded, and all the strands would have the same polarity. To hybridize with that DNA, the probe would need to have the complementary sequence and the opposite polarity. Before carrying out this experiment, Jennifer had no way of knowing which probe polarity would be required for hybridization. Since Jennifer did not understand the methods she was using, she made the mistake of using only RNA synthesized by the T7 RNA polymerase, and she was unlucky. The RNA synthesized by the T7 polymerase turned out to have the same sequence and polarity as the IMPORTANT gene in the M13 vector and therefore could not hybridize with it. If Jennifer had understood her methods, she would have prepared a probe mixture consisting of approximately equal amounts of radioactive RNA synthesized by the T7 and T3 RNA polymerases. Some of the radioactive RNA in such a mixture would have hybridized with the IMPORTANT gene in the M13 vector regardless of its polarity.

Some of you suggested that Jennifer's experimental problem was not the polarity of her probe but something else such as hybridization conditions. I gave partial credit to people who suggested a reasonable possible explanation for Jennifer's difficulties and who then proposed a way to correct those difficulties. However, I did not give full credit to such answers, because, regardless of whether Jennifer may have used incorrect hybridization conditions, she certainly should have employed probe RNA synthesized by both T3 and T7 RNA polymerases in her initial protocol.

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